Chlorophyll extract was measured as fluorescence and converted to concentration using spinach extract standards. Rock surface area was determined by water volume displacement ( Cooper and Testa, 2001) and epilithic algal biomass reported as μg Chl a cm−2 rock. Leaf material
was processed within a few days of collection to determine mass loss and fungal colonization from each stream site. Leaves were removed from each bag and gently rinsed with deionized water to remove sandy debris. From each leaf bag, ergosterol content (as an indication of fungal biomass) and organic leaf decay rates were determined. Ergosterol concentration (μg Ergosterol mg−1 ash-free dry weight (AFDW) leaf) was measured from 30 haphazardly collected hole punches of leaf tissue. Ergosterol was extracted from leaf punches by incubating in methanol for 2 h followed Apoptosis inhibitor by potassium hydroxide hydrolysis at 80 °C (Newell et al., 1988). Next, sterols were isolated through a pentane extraction at 21 °C. Pentane soluble sterol extracts were dried under a constant stream of N2 gas and re-dissolved in methanol for high pressure liquid chromatography (HPLC) analysis. The separation module (Waters 2695) injected 100 μl of solution through the column (Novapak C18) at a rate of 1.5 ml min−1. The Waters 2998 detector was set
at an absorbance of 282λ. Retention times and concentrations were compared to a pure ergosterol standard (Fluka HPLC grade > 95%; Newell et al., 1988). For leaf loss rates, leaves were dried in an oven at 60 °C until constant weight was reached. Leaf weights were corrected for the 30
this website hole punches taken for ergosterol. Dry leaves were ground and a subsample taken to determine AFDW (i.e., leaf organic content) by ashing in a muffle oven for 5 h at 550 °C. Sugar maple leaf decay rates (k) were calculated for each point using the negative natural log of the percent AFDW remaining at the end of the incubation ( Petersen and Cummins, 1974). Dissolved O2 and N2 concentrations from leaf incubations were determined using membrane inlet mass spectrometry (MIMS) from N2:Ar and O2:Ar ratios (Kana et al., 1994). Ar ratios were converted to concentrations using gas saturated water standards at 20 and 30 °C L-NAME HCl and by applying Henry’s law with published gas constants for Ar, N2, and O2 (Lide and Frederikse, 1995 and Wilhelm et al., 1977). O2 and N2 flux rates were calculated as the difference between initial and final gas concentrations divided by the incubation time. Leaf biofilm oxygen consumption (e.g., O2 uptake; R) and denitrification rates (e.g., N2 flux) were expressed as μg gas h−1 g−1 AFDW leaf. Prior to analysis, parameters were grouped as follows: (1) landscape, (2) water quality, (3) DOM characteristics, and (4) benthic. One N2 flux measurement was removed as an outlier prior to analysis because this point had a z-score < −4 (i.e., greater than 4 standard deviations way from the mean) and poor analytical reproducibility on multiple sample injections.